Project Summary Protein O-fucosyltransferase 2 (POFUT2) adds O-linked fucose to Thrombospondin Type 1 Repeats (TSRs) with the appropriate consensus sequence. TSRs are small cysteine-rich protein motifs that typically occur in tandem repeats and function in a wide variety of biologically essential contexts including cell attachment, migration, division, and apoptosis. The O-linked fucose on TSRs can be extended by a ?3-glucosyltransferase (B3GLCT) to form a Glucose?1-3Fucose disaccharide. The biological importance of these glycan modifications is highlighted by the fact that mice lacking POFUT2 display severe gastrulation defects, and human mutations in B3GLCT cause Peters Plus Syndrome (PPS), a congenital disorder of glycosylation with multiple developmental defects, many of which overlap with single gene defects in POFUT2 targets. POFUT2 only modifies folded TSRs, suggesting it can differentiate between folded and unfolded structures. Loss of POFUT2 blocks secretion of a growing number of the 49 POFUT2 target proteins in cell culture, and the effect appears more significant for proteins with tandem TSRs than singles. Recent data suggest that POFUT2 and B3GLCT work together in a novel non-canonical ER quality control system necessary for folding and secretion of POFUT2 targets. Addition of POFUT2 and GDP-fucose to an in vitro TSR folding reaction accelerates TSR folding by shifting the folding equilibrium towards the folded state, and addition of glucose by B3GLCT has an additional stabilizing effect. These observations have led to our main hypothesis, that the O-fucose glycan on an individual TSR stabilizes the folded state through interactions with specific residues of that TSR, accelerating the folding of nascent TSR-containing proteins in a sequential manner from N- to C-terminus in the ER. We will test this hypothesis in three Aims: First, we will use a newly developed Pofut2 conditional allele to determine whether TSR O-fucosylation is required in vivo for secretion and function of POFUT2 targets in the myeloid lineage and the developing limb mesenchyme. In Aim 2, we will analyze how O-fucosylation facilitates the folding of a single TSR by using NMR analysis to identify TSR residues in contact with the O- fucose glycan, and then introduce mutations in those residues to evaluate their importance in the stabilizing effects of the glycans. In Aim 3 we will examine whether tandem TSRs are more dependent on POFUT2 than a single TSR for secretion using a recently developed quantitative POFUT2-dependent secretion assay. Using this approach, we will determine whether the increased dependence of tandem TSRs on POFUT2 is an additive effect of stabilizing individual TSRs or if the glycan on one TSR cooperatively stabilizes an adjacent TSR, and whether O-fucosylation of tandem TSRs occurs sequentially during folding. Successful accomplishment of these aims will provide crucial molecular and cellular details regarding this novel quality control pathway necessary for a clear understanding of TSR folding, which could be used in the future to design therapies for treatment of diseases resulting from defects in the folding or function of POFUT2 targets.